Cicadas, or how I Am Such A Scientist, or a demonstration of good editing

Charles Q. Choi runs a bi-weekly series on the Guest Blog over at Scienctific American – Too Hard for Science? In these posts, he asks scientists about experiments that cannot be or should not be done, for a variety of reasons, though it would be fun and informative it such experiments could get done.

For one of his posts, he interviewed me. What I came up with, inspired by the emergence of periodic cicadas in my neighborhood, was a traditional circadian experiment applied to a much longer cycle of 13 or 17 years.

The idea: Everything in living organisms cycles. Some processes repeat in miliseconds, others in seconds, minutes or hours, yet others in days, months or years. Biological cycles that are most studied and best understood by science are those that repeat approximately once a day – circadian rhythms.

One of the reasons why daily rhythms are best understood is that pioneers of the field came up with a metaphor of the ‘biological clock‘ which, in turn, prompted them to adapt oscillator theory (the stuff you learned in school about the pendulum) from physics to biology.

These kinds of experiments – for example buildingPhase-ResponseCurves to a variety of environmental cues, or a variety of tests for photoperiodism (night-break protocol, skeleton photoperiods, resonance cycles, T-cycles, Nanda-Hamner protocol etc.) – take a long time to perform.

Each data point requires several weeks: measuring period and phase of the oscillation before and after the pulse (or a series of pulses) of an environmental cue in order to see how application of that cue at a particular phase of the cycle affects the biological rhythm (or the outcome of measuring daylength, e.g., reproductive response). It requires many data points, gathered from many individual organisms.

And all along the organisms need to be kept in constant conditions: not even the slightest fluctuations in light (usually constant darkness), temperature, air pressure, etc. are allowed.

It is not surprising that these kinds of experiments, though sometimes applied to shorter cycles (e.g., miliseconds-long brain cycles), are rarely applied to biological rhythms that are longer than a day, e.g., rhythms that evolved as adaptations to tidal, lunar and annual environmental cycles. It would take longer to do than a usual, five-year period of a grant, and some experiments may last an entire researcher’s career. Which is one of the reasons we know so little about these biological rhythms.

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Living out in the country, in the South, just outside Chapel Hill, NC, every day I open the door I hear the deafening and ominous-sounding noise (often described as “horror movie soundtrack) coming from the woods surrounding the neighborhood. The cicadas have emerged! The 13-year periodic cicadas, that is. Brood XIX.

Fortunately, once they emerge, cicadas are out for a few weeks, so my busy travel schedule did not prevent me from going to find them (just follow the sound) to take a few pictures and short videos.

There are three species of periodic cicadas that emerge every 17 years – Magicicada septendecim, Magicicada cassini and Magicicada septendecula. Each of these species has a ‘sister species’ that emerges every 13 years: M.tredecim, M. tredecassini and M.tredecula. A newer species split produced another 13-year species: Magicicada neotredecim. The species differ in morphology and color, while the 13 and 17-year pairs of sister species are essentially indistinguishable from each other. M.tredecim and M.neotredecim, since they appear at the same time and place, differ in the pitch of their songs: M.neotredecim sings a higher tone.

So, how do they count to 13 or 17?

While under ground, they undergo metamorphosis four times and thus go through five larval instars. The 13 and 17-year cicadas only differ in the duration of the fifth instar. They emerge simultaneously, live as adults for a few weeks, climb up the trees, sing, mate, lay eggs and die.

When the eggs hatch, the newly emerged larvae fall from the trees to the ground, dig themselves deeper down, latch onto the tree roots to feed on the sap, and wait another 13 or 17 years to emerge again.

There are a number of hypotheses (and speculations) why periodic cicadas emerge every 13 or 17 years, including some that home in on the fact that these two numbers are prime numbers (pdf).

Perhaps that is a way to fool predators which cannot evolve the same periodicity (but predators are there anyway, and will gladly gorge on these defenseless insects when they appear, whenever that is, even though it may not be so good for them). Perhaps this is a speciation mechanism, lowering the risk of hybridization between recently split sister species?

Or perhaps that is all just crude adaptationist thinking and the strangeness of the prime-number cycles is in the eye of the beholder – the humans! After all, if an insect shows up every year, it is not very exciting. Numerous species of annual cicadas do that every year and it seems to be a perfectly adaptive strategy for them. But if an insect, especially one that is so large, noisy and numerous, shows up very rarely, this is an event that will get your attention.

Perhaps our fascination with them is due to their geographic distribution. Annual cicadas may also have very long developmental times, but all of their broods are in one place, thus the insects show up every year. In periodic cicadas, different broods appear in different parts of the country, which makes their appearance rare and unusual in each geographic spot.

In any case, I am more interested in the precision of their timing than in potential adaptive explanations for it. How do they get to be so exact? Is this just a by-product of their developmental biology? Is 13 or 17 years just a simple addition of the duration of five larval stages?

Or should we consider this cycle to be an output of a “clock” (or “calendar”) of sorts? Or perhaps a result of interactions between two or more biological timepieces, similarly to photoperiodism? In which case, we should use the experimental protocols from circadian research and apply them to cicada cycles.

Finally, it is possible that a ling developmental cycle is driven by one timing mechanism, but the synchronization of emergence in the last year is driven by another, perhaps some kind of clock that may be sensitive to sound made by other insects of the same species as they start digging their way up to the surface.The problem: In order to apply the standard experiments (like construction of a Phase-Response Curve, or T-cycles), we need to bring the cicadas into the lab. And that is really difficult to do. Husbandry has been a big problem for research on these insect, which is why almost all of it was done out in the field.

When kept in the lab, the only way to feed them is to provide them with the trees so they can drink the sap from the roots. This makes it impossible to keep them in constant conditions – trees require light and will have their own rhythms that the cicadas can potentially pick up, as timing cues, from the sap. So, the first thing we need to do is figure out a way to feed them artificially, without reliance on living trees for food.

Also, we do not know which environmental cues are relevant. Is it light cycle? Photoperiod? Or something cycling in the tree-sap? Or temperature cycles? What are the roles of developmental hormones like Juvenile Hormone or Ecdysone? We would have to test all of them simultaneously, hoping that at least one of them turns out to be the correct one.

Second, more obvious problem, is time. These experiments would last hundreds of years, perhaps thousands! Some experiments rely on outcomes of previous experiments for the proper design. Who would do them? What funding agency would finance them? Why would anyone start such experiments while knowing full well that the results would not be known within one’s lifetime? Isn’t this too tantalizing for a scientist’s curiosity?

The solution? One obvious solution is to figure out ways to get to the same answers in shorter time-frames. Perhaps by sequencing the genome and figuring out what each gene does (perhaps by looking at equivalents in other species, like fruitflies, or inserting them into Drosophila and observing their effects), hoping to find out the way timing is regulated. This will probably not answer all our questions, but may be good enough.

Another way is to set aside space and funding for such experiments and place them into an unusual administrative framework – a longitudinal study guided by an organization, not a single researcher getting a grant to do this in his or her lab. This way the work will probably get done, and the papers will get published somewhere around 2835 A.D.

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See? How long and complex my text is? Now go back to the post by Charles to see again how nicely he edited the story.